Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (k(cat)). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5-1.7 mu m/s; 25 degrees C) in the in vitro motility assay as well as maximum actin activated catalytic activity (k(cat); 8-9 s(-1)) and actin concentration for half maximal activity (K-ATPase; 70-80 mu M) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.

The eukaryotic translation initiation factor 4-gamma (eIF4G) is important for the initiation of protein synthesis and phosphorylation on S1108 regulates this function of eIF4G. Thus, increased phosphorylation has been reported in conditions associated with increased protein synthesis such as meal feeding and insulin/IGF-1 treatment whereas decreased phosphorylation occurs following starvation, dexamethasone treatment, in sepsis and in atrophic denervated hind-limb muscle. The aim of the present study was to test the hypothesis that S1108 phosphorylation of eIF4G is differentially affected in denervated atrophic hind-limb muscles and denervated hypertrophic hemidiaphragm muscle. Protein expression and phosphorylation in innervated and 6-days denervated atrophic hind-limb muscles (pooled gastrocnemius and soleus) and hypertrophic hemidiaphragms were studied semi-quantitatively using Western blots. Total expression of eIF4G did not change in denervated hind-limb muscles but increased about 77% in denervated hemidiaphragm. S1108 phosphorylated eIF4G decreased about 64% in denervated hind-limb muscles but increased about 1.3-fold in denervated hemidiaphragm. The ratio of S1108 phosphorylated eIF4G to total eIF4G decreased about 60% in denervated hind-limb muscles but no statistically significant change was observed in denervated hemidiaphragm. The differential effect of denervation on eIF4G expression and S1108 phosphorylation in hemidiaphragm (hypertrophic) and hind-limb muscle (atrophic) may represent a regulatory mechanism that helps clarify the differential response of these muscles following denervation.

Background: Forkhead box O (FoxO) transcription factors and E3 ubiquitin ligases such as Muscle RING finger 1 (MuRF1) are believed to participate in the regulation of skeletal muscle mass. The function of FoxO transcription factors is regulated by post-translational modifications such as phosphorylation and acetylation. In the present study FoxO1 protein expression, phosphorylation and acetylation as well as MuRF1 protein expression, were examined in atrophic and hypertrophic denervated skeletal muscle. Methods: Protein expression, phosphorylation and acetylation were studied semi-quantitatively using Western blots. Muscles studied were 6-days denervated mouse hind-limb muscles (anterior tibial as well as pooled gastrocnemius and soleus muscles, all atrophic), 6-days denervated mouse hemidiaphragm muscles (hypertrophic) and innervated control muscles. Total muscle homogenates were used as well as separated nuclear and cytosolic fractions of innervated and 6-days denervated anterior tibial and hemidiaphragm muscles. Results: Expression of FoxO1 and MuRF1 proteins increased 0.3-3.7-fold in all 6-days denervated muscles studied, atrophic as well as hypertrophic. Phosphorylation of FoxO1 at S256 increased about 0.8-1-fold after denervation in pooled gastrocnemius and soleus muscles and in hemidiaphragm but not in unfractionated anterior tibial muscle. A small (0.2-fold) but statistically significant increase in FoxO1 phosphorylation was, however, observed in cytosolic fractions of denervated anterior tibial muscle. A statistically significant increase in FoxO1 acetylation (0.8-fold) was observed only in denervated anterior tibial muscle. Increases in total FoxO1 and in phosphorylated FoxO1 were only seen in cytosolic fractions of denervated atrophic anterior tibial muscle whereas in denervated hypertrophic hemidiaphragm both total FoxO1 and phosphorylated FoxO1 increased in cytosolic as well as in nuclear fractions. MuRF1 protein expression increased in cytosolic as well as in nuclear fractions of both denervated atrophic anterior tibial muscle and denervated hypertrophic hemidiaphragm muscle. Conclusions: Increased expression of FoxO1 and MuRF1 in denervated muscles (atrophic as well as hypertrophic) suggests that these proteins participate in the tissue remodelling occurring after denervation. The effect of denervation on the level of phosphorylated and acetylated FoxO1 differed in the muscles studied and may be related to differences in fiber type composition of the muscles.

BACKGROUND: p38 mitogen-activated protein kinase has been implicated in both skeletal muscle atrophy and hypertrophy. T317 phosphorylation of the p38 substrate mitogen-activated protein kinase-activated protein kinase 2 (MK2) correlates with muscle weight in atrophic and hypertrophic denervated muscle and may influence the nuclear and cytoplasmic distribution of p38 and/or MK2. The present study investigates expression and phosphorylation of p38, MK2 and related proteins in cytosolic and nuclear fractions from atrophic and hypertrophic 6-days denervated skeletal muscles compared to innervated controls.

METHODS: Expression and phosphorylation of p38, MK2, Hsp25 (heat shock protein25rodent/27human, Hsp25/27) and Hsp70 protein expression were studied semi-quantitatively using Western blots with separated nuclear and cytosolic fractions from innervated and denervated hypertrophic hemidiaphragm and atrophic anterior tibial muscles. Unfractionated innervated and denervated atrophic pooled gastrocnemius and soleus muscles were also studied.

RESULTS: No support was obtained for a differential nuclear/cytosolic localization of p38 or MK2 in denervated hypertrophic and atrophic muscle. The differential effect of denervation on T317 phosphorylation of MK2 in denervated hypertrophic and atrophic muscle was not reflected in p38 phosphorylation nor in the phosphorylation of the MK2 substrate Hsp25. Hsp25 phosphorylation increased 3-30-fold in all denervated muscles studied. The expression of Hsp70 increased 3-5-fold only in denervated hypertrophic muscles.

CONCLUSIONS: The study confirms a differential response of MK2 T317 phosphorylation in denervated hypertrophic and atrophic muscles and suggests that Hsp70 may be important for this. Increased Hsp25 phosphorylation in all denervated muscles studied indicates a role for factors other than MK2 in the regulation of this phosphorylation.

Purpose Socioeconomic factors have been suggested to influence the prescribing of newer and more expensive drugs. In the present study, individual and health care provider factors were studied in relation to the prevalence of differently priced drugs. Methods Register data for dispensed drugs were retrieved for 18486 individuals in a county council in Sweden. The prevalence of dispensed drugs was combined with data for the individual's gender, age, education, income, foreign background, and type of caregiver. For each of the diagnostic groups (chronic obstructive pulmonary disease [COPD], depression, diabetes, and osteoporosis), selected drugs were dichotomized into cost categories, lower and higher price levels. Univariate and multivariate logistic regressions were performed using cost category as the dependent variable and the individual and provider factors as independent variables. Results In all four diagnostic groups, differences were observed in the prescription of drugs of lower and higher price levels with regard to the different factors studied. Age and gender affected the prescription of drugs of lower and higher price levels more generally, except for gender in the osteoporosis group. Income, education, foreign background, and type of caregiver affected prescribing patterns but in different ways for the different diagnostic groups. Conclusions Certain individual and provider factors appear to influence the prescribing of drugs of different price levels. Because the average price for the cheaper drugs versus more costly drugs in each diagnostic group was between 19% and 69%, there is a risk that factors other than medical needs are influencing the choice of drug. Copyright (c) 2013 John Wiley & Sons, Ltd.

ABSTRACT: BACKGROUND: The present study examines the hypothesis that Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signaling is increased in hypertrophic and decreased in atrophic denervated muscle. Protein expression and phosphorylation of Akt1, Akt2, glycogen synthase kinase-3beta (GSK-3beta), eukaryotic initiation factor 4E binding protein 1 (4EBP1), 70 kD ribosomal protein S6 kinase (p70S6K1) and ribosomal protein S6 (rpS6) were examined in six-days denervated mouse anterior tibial (atrophic) and hemidiaphragm (hypertrophic) muscles. RESULTS: In denervated hypertrophic muscle expression of total Akt1, Akt2, GSK-3beta, p70S6K1 and rpS6 proteins increased 2-10 fold whereas total 4EBP1 protein remained unaltered. In denervated atrophic muscle Akt1 and Akt2 total protein increased 2-16 fold. A small increase in expression of total rpS6 protein was also observed with no apparent changes in levels of total GSK-3beta, 4EBP1 or p70S6K1 proteins. The level of phosphorylated proteins increased 3-13 fold for all the proteins in hypertrophic denervated muscle. No significant changes in phosphorylated Akt1 or GSK-3beta were detected in atrophic denervated muscle. The phosphorylation levels of Akt2, 4EBP1, p70S6K1 and rpS6 were increased 2-18 fold in atrophic denervated muscle. CONCLUSIONS: The results are consistent with increased Akt/mTOR signaling in hypertrophic skeletal muscle. Decreased levels of phosphorylated Akt (S473/S474) were not observed in denervated atrophic muscle and results downstream of mTOR indicate increased protein synthesis in denervated atrophic anterior tibial muscle as well as in denervated hypertrophic hemidiaphragm muscle. Increased protein degradation, rather than decreased protein synthesis, is likely to be responsible for the loss of muscle mass in denervated atrophic muscles.

Background: The purpose of this study was to investigate awareness among nurses regarding their new role as reporters of adverse drug reactions in Sweden and factors that may influence reporting by nurses.

Methods: In 2007, all nurses were included in the adverse drug reaction reporting scheme in Sweden. A questionnaire was sent to 753 randomly selected nurses in September 2010.

Results: Of the 453 (60%) responding nurses, 265 (58%) were aware that nurses were included in the reporting of adverse drug reactions. Sixty-one nurses (14%) stated that they had reported an adverse drug reaction. Fifteen percent (n = 70) of the respondents had received training about reporting of adverse drug reactions. Almost one third of these (n = 21, 30%) had reported an adverse drug reaction on at least one occasion. Among nurses without training, a smaller proportion (n = 40, 11%, P < 0.05) had reported an adverse drug reaction on at least one occasion. The two factors considered most important by nurses for reporting were the severity of the adverse drug reaction and if the reaction was to a newly approved drug. A majority of the nurses (n = 397, 88%) were interested in a training course in pharmacology as part of their ongoing professional development. One third (32%) of all nurses stated that one reason for not reporting a suspected adverse drug reaction was that the physician responsible did not regard the reaction necessary to report.

Conclusion: We found that more than half of the study population of nurses in Sweden were aware of their new role as reporters of adverse drug reactions, but few of the responding nurses had reported an adverse drug reaction. Given that training seems to be associated with high reporting frequency, we suggest more training in pharmacovigilance for nurses.